Faculty Profile

Michael K. Reedy, MD (University of Washington) Professor, Department of Cell Biology Programs: CMB, Structural Biology and Biophysics	Among known muscles, none show greater macromolecular order than certain insect flight muscles (IFM). Our favorite IFM is that of giant Lethocerus waterbugs. Even after permeabilization, glycerination, and months to years of storage at -80°C, its near-crystalline lattices of myofilaments and crossbridges preserve unimpaired mechanical and structural responses in (and transitions between) a multitude of physiological and pharmacological states. With collaboration from five outside labs, we are currently pursuing a 3-way approach to clarify individual motor actions and integrated ensemble performance of myosin crossbridges, by combining X-ray diffraction and quick-freezing for electron microscopy with mechanical (tension) monitoring of muscle fibers in contraction and related states. Synchrotron-source fiber X-ray patterns from exposures lasting 0.1 to 100 msec correlate closely with computed image Fourier transforms of thin-section electron micrographs (EMs) from fibers quick-frozen in 0.1-1 ms. 3-D EM tomography provides density maps of quick-frozen crossbridges into which molecular models of actin and myosin are fitted to discover the pre-force (weakly bound) and active working-stroke conformations and molecular choreography of these interacting molecules. Structural responses to sudden changes in length or temperature are especially informative. High-resolution cryo-X-ray diffraction (to ~7Å) of fibers rapidly and homogeneously frozen, and computer modeling to simulate experimental X-ray patterns round out our approach. Present funding is assured through July 2011.

E-mail mike.reedy@cellbio.duke.edu 458 Sands Bldg., Box 3011 Duke University Medical Center Durham, NC 27710 Telephone 919-684-5674 Fax 919-681-9929

Selected Publications

AL-Khayat, H. A., Hudson, L., Reedy, M. K., Irving, T. C. & Squire, J. M. (2004). Modelling oriented macromolecular assemblies from low-angle x-ray fibre diffraction data with the program MOVIE: Insect flight muscle as an example. Fibre Diffraction Review 12:50-60. -PDF-

Liu, J., Reedy, M. C., Goldman, Y. E., Franzini-Armstrong, C., Sasaki, H., Tregear, R. T., Lucaveche, C., Winkler, H., Baumann, B. A. J., Squire, J. M., Irving, T. C., Reedy, M. K. & Taylor, K. A. (2004). Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges. Journal of Structural Biology 147:268-282. -PDF-

Tregear, R. T., Reedy, M. C., Goldman, Y. E., Taylor, K. A., Winkler, H., Franzini-Armstrong, C., Sasaki, H., Lucaveche, C. & Reedy, M. K. (2004). Cross-bridge number, position and angle in target zones of cryofixed isometrically active insect flight muscle. Biophysical Journal 86:3009-3019. -PDF-

Linari, M., Reedy, M. K., Reedy, M. C., Lombardi, V. & Piazzesi, G. (2004). Ca-Activation and stretch-activation in insect flight muscle. Biophysical Journal 86:1101-1111. -PDF-

Reedy, M. C. (2005). 3-D structure of myosin crossbridges in insect flight muscle: Toward visualization of the conformations during myosin motor action. In Nature's Versatile Engine: Insect Flight Muscle Inside and Out (Vigoreaux, J. O., ed.). Landes BioScience, Georgetown, TX.
(Available by on-line subscription at http://www.eurekah.com/isbn.php?isbn=1-58706-279-8&chapid=2444&bookid=131&catid=20)

Squire, J. M., AL-Khayat, H. A., Harford, J. J., Hudson, L., Irving, T. C., Knupp, C. & Reedy, M. K. (2003). Modelling muscle motor conformations using low-angle X-ray diffraction. IEE Proceedings Nanobiotechnology 150:103-110. -PDF-

AL-Khayat, H. A., Hudson, L., Reedy, M. K., Irving, T. C. & Squire, J. M. (2003). Myosin head configuration in relaxed insect flight muscle: X-ray modelled resting crossbridges in a pre-powerstroke state are poised for actin binding. Biophys J 85:1063-1079. -PDF-

Lewis, M. K., Nahirney, P. C., Chen, V., Adhikari, B. B., Wright, J., Reedy, M. K., Bass, A. H. & Wang, K. (2003). Concentric intermediate filament lattice links to specialized Z-band junctional complexes in sonic muscle fibers of the type I male midshipman fish. J Struct Biol 143:56-71. -PDF-

Chen, L. F., Winkler, H., Reedy, M. K., Reedy, M. C. & Taylor, K. A. (2002). Molecular modeling of averaged rigor crossbridges from tomograms of insect flight muscle. J Struct Biol 138:92-104. -PDF-

Reedy, M. C., Bullard, B. & Vigoreaux, J. O. (2000). Flightin is essential for thick filament assembly and sarcomere stability in Drosophila flight muscles. J.Cell Biol. 151:1483-1500. -PDF-

Reedy, M. C. (2000). Visualizing myosin's power stroke in muscle contraction. J Cell Sci 113:3551-3562. -PDF-

Taylor, K. A., Schmitz, H., Reedy, M. C., Goldman, Y. E., Franzini-Armstrong, C., Sasaki, H., Tregear, R. T., Poole, K. J. V., Lucaveche, C., Edwards, R. J., Chen, L. F., Winkler, H. and Reedy, M. K. (1999) Tomographic 3-D reconstruction of quick frozen, Ca2+-activated contracting insect flight muscle. Cell. 99:421-31. -PDF-

Tregear, R. T., Edwards, R. J., Irving, T. C., Poole, K. J. V., Reedy, M. C., Schmitz, H., Towns-Andrews, E. and Reedy, M. K. (1998) X-ray diffraction indicates that active crossbridges bind to actin target zones in insect flight muscle. Biophys.J. 74:1439-1451. -PDF-

Reedy, M. C., Reedy, M. K., Leonard, K. and Bullard, B. (1994) Gold-Fab immuno-electron-microscopic localization of troponin H and troponin T in Lethocerus flight muscle. J. Mol. Biol. 239: 52-67.

Reedy, M. C. and Beall, C. (1993) Ultrastructure of developing flight muscle in Drosophila. I. Assembly of myofibrils; II. Formation of the myotendon junction. Dev. Biol. 160: 443-479.

Taylor, K. A., Reedy, M. C., Córdova, L. and Reedy, M. K. (1989) Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer; II. The rigor actin layer. J. Cell Biol. 109: 1085-1102; 1103-1123.

Current Projects
Michael Reedy (lab chief): Structure & action of crossbridges & myofilaments in IFM of giant waterbugs. Synchrotron X-ray diffraction and quick-freeze EMy, seeking snapshots of the crossbridge power-stroke in contracting IFM. Also seeking to improve X-ray and thin-section EM resolution from 4.0 to 1.0 nm by developing new cryo-methods.

Mary Reedy (Research Associate): Ultrathin-section EM analysis of IFM crossbridges in diverse physiological states. Develop collaborations on muscle and cytoskeletal ultrastructure wherein our lab's methods can benefit related research topics.

Carmen Lucaveche (Research Associate): Ultrathin-section analysis of IFM crossbridges. Snapshots of the powerstroke. Optimizing chemical and cryofixation of myofibrils, myofilaments & crossbridges.

With Ken Taylor's lab (Florida State Univ, Tallahassee): 3D computer image analysis of crossbridge structure & action, especially EM tomography at molecular resolution from multiple EM tilt views.

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